Amplified Insert Assembly is a method of "BioBricking™" two biological parts (i.e. pieces of DNA) together. For more information on BioBricking see [http://partsregistry.org/Help:Contents this link]. A You-Tube [http://www.youtube.ug/watch?v=Tvxx2bL0R2I&context=C3df62b2ADOEgsToPDskIOrrbFVhSy4joiAlQXxIYp video summary of the Amplified Insert Assembly method] provides a quick visual introduction.

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Amplified Insert Assembly is a method of "BioBricking" two biological parts (i.e. pieces of DNA) together and was developed by [[User:Michael A. Speer|Mike Speer]] and Dr. Tom Richard. For more information on bio-bricking see [http://partsregistry.org/Help:Contents this link]. This method combines the ease and speed of 3A assembly with the reliability of standard assembly. In comparison to [[Synthetic Biology:BioBricks/3A assembly|3A assembly]], this method can take up to two hours longer; however, the actual additional time spent at the bench is minimal. Major benefits of this assembly method over other bio-brick assembly methods include:

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This method combines the ease and flexibility of [[Synthetic Biology:BioBricks/3A assembly|3A assembly]] with the reliability of [[BioBricks construction tutorial|standard assembly]]. In comparison to 3A assembly, this method can take up to two hours longer; however, the additional time spent at the bench is minimal. Major benefits of this assembly method over other BioBrick™ assembly methods include:

*No need to order [[In-fusion biobrick assembly|custom oligos for each assembly]].

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*Really low background (99% of colonies are correct)

**This means less sequencing

**This means less sequencing

*Easy transformation (use homemade competent cells)

*Easy transformation (use homemade competent cells)

*Less culturing

*Less culturing

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**This is because one plasmid prep can supply many PCR inserts. So common parts (i.e. promoters and RBSs) can be used over and over again.

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**This is because one plasmid prep can supply many PCR inserts. So minipreps of common parts (i.e. promoters and RBSs) can be used over and over again.

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This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:

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This protocol is typically used to do BioBrick™ assembly with restriction sites in the [[Biobrick standard| RFC 10 BioBrick Standard ]]:<br>

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<center>

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<center>'''-----EcoRI--XbaI--Part--SpeI--PstI-----'''</center>

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'''-----EcoRI--XbaI--''Part''--SpeI--PstI-----'''<br>

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</center>

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But it can also be applied to [[The BioBricks Foundation:Standards/Technical/Formats|any other format]] in which the two inner sites form an inactive "scar" and the two outer sites can be heat inactivated.

The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids. The eventual goal of assembly is to get these parts on the same plasmid next to one another.

The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids. The eventual goal of assembly is to get these parts on the same plasmid next to one another.

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This is a consensus protocol. For more specific lab protocols see the pages below

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'''This is a consensus protocol. For more information see the pages below'''

3. [[Engineering BioBrick vectors from BioBrick parts/Restriction digest|Digest]] the "vector" with the [[Enzyme selection for BioBricks digest|appropriate restriction endonucleases]] for 2 hours.(do this while while PCR is running)<br>

4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]].<br>

4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]].<br>

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5. [[Richard Lab:Restriction Digest|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>

This method combines the ease and flexibility of 3A assembly with the reliability of standard assembly. In comparison to 3A assembly, this method can take up to two hours longer; however, the additional time spent at the bench is minimal. Major benefits of this assembly method over other BioBrick™ assembly methods include:

But it can also be applied to any other format in which the two inner sites form an inactive "scar" and the two outer sites can be heat inactivated.

The two parts you want to assemble will be labeled "insert" and "vector" and will be initially contained on separate plasmids. The eventual goal of assembly is to get these parts on the same plasmid next to one another.

This is a consensus protocol. For more information see the pages below

6. Add 6μL Antarctic Phosophatase Buffer and 1μL Antarctic Phosphatase to the "vector" digest and incubate until the "insert" digest is done.
7. Kill all reactions by incubating for 20 mins at 80°C.
8. Ligate at a molar ratio of 4:1 (insert:vector).
9. Transform your cells.
10. Plate the transformed cells on plates with the same antibiotic as the "vector" resistance.
11. Celebrate.

If you already have PCR insert ready to go (i.e. you ran the PCR the night before from old miniprep) then it only takes about 4 hours.

Notes

The DpnI eliminates any background from the insert PCR.

The phosphatase eliminates any background vector.

The "vector" will be digested for a total of thee hours (including nearly one hour with Antarctic Phosphatase)

The "insert" will only be digested for one hour. This is okay as there is a lot of it.

Detractors of this method may say that it's risky to PCR the inserts because of mutations. We say: